Co-Purification of Dihydrofolate Synthetase and N10Formyltetrahydropteroyldiglutamate Synthetase from E. Coli

  • Robert Ferone
  • Alice Warskow
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 163)

Abstract

The enzymatic activities which add a single glutamate to di-hydropteroate (H2Pte) and to N10 formyltetrahydropteroylglutamate (N10formyl H4PteGlu) remained at a constant ratio when various purification techniques were used, including ammonium sulfate fractionation, isoelectric focusing, Polyacrylamide gel electrophoresis, and column chromatography on Sephadex G-100, DEAE-Sephadex, CM-Sephadex, ADP-Sepharose, N10formylfolate-agarose or matrix Gel Red-A. The best combination of these methods yielded 900-fold purified enzyme. However, the kinetic properties were dependent upon the substrate used. H2Pte was a noncompetitive inhibitor (Kii = 1.1 µM) of the utilization of N10formylHi,PteGlu, but no inhibition was detected in the reciprocal experiment. Aminopterin was a competitive inhibitor (Kis = 370 µM) of the reaction with N10formylH4PteGlu but was not inhibitory with H2Pte as substrate. In the dihydrofolate synthetase reaction, in Tris-HCl, pH 8.9 with 50 mM KCl, the apparent Km values for glutamate and MgATP were 3.5 mM and 8.1 µM respectively. With N10formylH4PteGlu as substrate, these Km values were 1.2 mM and 80 µM. Since the two activities were not separated by protein purification procedures but exhibited different kinetic properties (including lack of reciprocal inhibition), the data suggest these reactions are catalyzed on independent sites of a common protein.

Keywords

Synthetase Activity Initial Final Major Protein Band Parallel 1anes Enzyme Reaction Mixture 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Springer Science+Business Media New York 1983

Authors and Affiliations

  • Robert Ferone
    • 1
  • Alice Warskow
    • 1
  1. 1.Department of MicrobiologyWellcome Research LaboratoriesResearch Triangle ParkUSA

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